A number of diagnostic assays are carried out in automated equipment using multiwell plastic plates and automated equipment in which a vertical beam of light is used in making spectrophotometric readings in the individual wells of the plates. These plates have several common features: plastic wells with optically transparent bottom is isolated from one another with respect to liquid contained therein but physically connected in a precise geometric pattern. The wells are typically part of a plastic carrier plate, and the automated equipment is designed to have a movable stage into which one or more multiwell plates precisely fit. Most commonly these multiwell plates contain 96 wells arranged in an 8.times.12 pattern, although plates containing other numbers of wells are also available.
One common use of multiwell plates is in an automated diagnostic assay using antibodies to bind an analyte in a sample added to one or more of the wells of the plate. Before a multiwell plate can be used for this type of test, it must be coated with the appropriate antibody. This is normally accomplished by the user and consists of adding an antibody solution to the individual wells, followed by incubating and removing excess solution. During the incubation interval, the antibody binds non-convalently to the wall and bottom of the individual wells. The amount of antibody and the tenacity of the bond that the antibody makes to the walls of the individual wells are important factors in the sensitivity and reliability of the diagnostic test that uses the multiwell plate.
When antibody-coated plates are used in an automated, vertical beam spectrophotometer, samples are added to the individual wells. The plate is then placed in the movable stage of the spectrophotometer. Activating the machine causes the stage to automatically advance into the machine, and a series of preprogrammed steps occur. In a number of machines, hollow needles descend into some or all of the wells and either inject a liquid containing reagents used in the assay or remove a liquid from a previous step. The stage then shifts sufficiently to allow the process to be repeated in the next group of wells. After the last chemical step of the sequence, which typically results in the formation of a colored product, the stage shifts to a new location so that the individual wells are placed in proper register either above or below a light source which passes a beam of light vertically through the well to a detector which measures the amount of transmitted light of a particular wavelength. This reading is converted automatically to a reading of the amount of analyte present in the sample, since the amount of color formed in the reaction is related to the amount of analyte.
The chemical and biochemical reactions that eventually result in color formation take place at the surfaces of the individual wells. Specifically, it is the surface area of the well wetted by the antibody solution initially used to coat the wells that sets the maximum level of antibody which can be bound. Since the geometry of the individual wells is essentially fixed by the constraints of the automated equipment, there is a practical limit to antibody adsorption on typical multiwell plates in current use. This can cause falsely low readings when large amounts of analyte are present, since not enough antibody will be present on the well walls to bind all of the analyte, as well as problems in sensitivity.
One attempt to overcome this limitation has employed porous latex beads contained in the wells. The antibody is bound to the latex, and the well simply becomes a chamber containing the beads.
While this approach does provide a significant increase in bound antibody, it suffers from serious practical problems. For example, the beads are typically unconstrained and can be removed accidently during the filling and emptying cycles in the automated equipment. Tests utilizing beads are therefore more sensitive to slight variations in machine fill and empty cycles than are multiwell plates that do not contain beads.
A second problem with current multiwell devices relates to the tenacity of antibody binding to well walls. Since the adsorption of antibody is basically passive (i.e., hydrophobic) in current multiwell plates, slight differences in surface characteristics from well to well can provide significant differences in the amount of antibody bound. These variations can significantly effect the reliability of diagnostic assays that utilize antibody-coated multiwell plates. Although the use of antibodies bound to latex beads avoids this problem, the latex beads are subject to the problems discussed above.
Accordingly, there remains a need for improvements in multiwell plates to provide for increased antibody binding in a more reliable manner.